Oligonucleotide synthesis has been found to be extremely useful in making primers for polymerase chain reaction (PCR) replication. In particular, oligonucleotides can be tailored to bind to a particular region of complementary DNA thereby allowing specific segments of DNA to be amplified. This aspect of oligonucleotides has created an interest in using oligonucleotides in therapeutic and diagnostic applications, and specifically for use in modifying gene and protein function in a sequence specific manner. For these and other applications, oligonucleotides must be produced in large quantities and be of high purity.
DNA and RNA oligonucleotide synthesis involves the bonding of multiple nucleosides in series. This process generally requires the steps of (1) the de-blocking of the 5′-hydroxyl group on a first nucleoside, nucleotide, or oligonucleotide; (2) activation of a phosphoramidite monomer base; (3) binding the first activated nucleoside to the activated phosphoramidite via phosphite linkage; and (4) oxidizing the phosphite linkage to form a more stable phosphate linkage. These four steps can be repeated until the desired oligonucleotide is produced.
Phosphoramidites are, therefore, important as building blocks in the synthesis of oligonucleotides. Phosphoramidites for a variety of nucleosides are commercially available through a variety of vendors. 3′-O-phosphoramidites are the most widely used amidites, but the synthesis of oligonucleotides can also involve 5′-O- and 2′-O-protected phosphoramidites (Wagner et al., Nucleosides & Nucleotides, 1997, 17, 1657-1660; Bhan et al., Nucleosides & Nucleotides, 1997, 17, 1195-1199). There are also many phosphoramidites available that are not nucleosides (Cruachem Inc., Dulles, Va.; Clontech, Palo Alto, Calif.).
As indicated above, oligonucleotide synthesis typically involves the coupling of a 3′-O-phosphoramidite to a 5′-OH group of a nucleoside, nucleotide, or oligonucleotide. One of the steps in this synthesis process is the activation of the phosphoramidite which is achieved by cleaving off one of the groups protecting the phosphorous linkage. The resulting activated phosphorous is then able to bond to the active 5′-hydroxyl group of the nucleoside base. An example of this reaction is shown in the scheme below wherein a phosphoramidite (I) is reacted with an oligonucleoside (II) bound to the primer support in the presence of a phosphoramidite activator to form an internucleoside linkage (III):
In this example, R is moiety such as dimethoxytrityl (DMT), 2′-O-(tert-butyl)-dimethylsilyl (TBDMS), 2′-O-[(triisopropyl-silyl)oxy]methyl (TOM), oligonucleotides and analogs thereof, or the like; Pg is a phosphorous protecting group such as alkyl, —CH2CH2CN, —CH2CH═CHCH2CN, para-CH2C6H4CH2CN, —(CH2)2-5N(H)COCF3, —CH2CH2Si(C6H5)2CH3, CH2CH2N(CH3)COCF3, and the like; X and X′ are independently hydrogen, fluoro, alkoxy, —O-tert-butyldimethyl silyl (OTBDMS), —O-methoxy methyl (OMOM), 2′-O-methoxyethyl (2′-O-MOE), or the like; and B and B′ are independently a moiety derived from adenine, cytosine, guanine, thymine, or uracil.
Oligonucleotides formed via the above-mentioned scheme are known in the art, see e.g. Rüdiger Welz and Sabine Müller, “5-Benzylmercapto-1H-tetrazole as activator for 2′O-TBDMS phosphoramidite building blocks in RNA synthesis”, Tetrahedron Letters 43, 2002, p. 795-97. To supply the growing demand for these oligonucleotides, there is a desire to improve the synthesis of oligonucleotides on a commercial scale (Noe, Kaufhold, New Trends in Synthetic Medicinal Chemistry, Wiley-VCh Weinheim, 2000, 261). To this end, much effort has been expended in developing phosphoramidite activators.
The first activator described for phosphoramidite chemistry was 1H-tetrazole. Subsequently, more potent activators have been developed including 5-methylthio-1H-tetrazole, 5-nitrophenyl-1H-tetrazole, 5-ethylthio-1H-tetrazole, and 4,5-dicyanoimidazole. More recently, 5-benzylmercaptotetrazole (BMT) (also known as benzylthiotetrazole (BTT)) was introduced as an ideal activator for certain RNA synthesis techniques such as activation of TOM-protected and TBDMS-protected RNA phosphoramidites. See e.g. X. Wu and S. Pitsch, Nucleic Acitds Research, 1998, 26, 4315-23); S. Pitsch, et al., Helv. Chim. Acta, 2001, 84, 3773-3795; and R. Welz and S. Müller, “5-Benzylmercapto-1H-tetrazole as activator for 2′O-TBDMS phosphoramidite building blocks in RNA synthesis”, Tetrahedron Letters 43, 2002, p. 795-97.
BMT allows for efficient RNA synthesis with as much as 50% less TBDMS or TOM monomer as compared to that required by processes using 1H-tetrazole. In addition, BMT has a higher solubility at lower temperatures than does 1H-tetrazole and BMT does not crystallize or clog lines below 19° C. However, it has been reported that BMT's maximum solubility in acetonitrile is about 0.33M (see http://www.eurogentec.be/code/EN/what.asp?pk_id_what=85) and the hydrophobic nature of BMT has limited its commercial use in acetonitrile to solutions less than or equal to 0.25M. Above that concentration, the BMT in acetonitrile becomes unstable and tends to clog reagent lines in commercial operations. As a result, commercial use of BMT in acetonitrile have been limited to concentrations less than or equal to 0.25M although solutions above 0.3M could advantageously lead to more efficient and rapid activation and less phosphoramidite waste. In addition, BMT in acetonitrile above 0.3M is safer and not explosive and, therefore, can be made in large quantities and stored and shipped in metal containers, such as 200L stainless steel pressure dispensing systems.
Applicant have overcome these and other shortcomings of the prior art with the present invention.